Neuropathic pain has been defined as “a pain that occurs as a direct result of an injury or a disease that directly affects the somatosensory system” (Treede, R. D., Jensen, T. S., Campbell, J. N., et al., 2008). This type of pain differs from nociceptive, somatic or visceral pain, because the latter occurs in non-nerve tissue and is caused either by a mechanical injury or damage caused internally by some pathology. Nociceptive pain is usually associated with an inflammatory process following tissue damage, and as a result reversible adaptive changes occur in the sensory nervous system. This leads to hypersensibility to pain, which is a protective mechanism that alerts and prevents subsequent damage at the site of injury, ensuring adequate repair of the damaged tissue. This painful sensation is mediated in the periphery by primary sensory neurons of high threshold, the so-called nociceptors, which transmit information to the brain through nociceptive pathways of the spinal cord. In this case, neither the structure nor the function of the nervous system is damaged, and the pain disappears when the damaged tissue has been repaired. Unlike this mechanism of damage→pain/inflammation→tissue repair→absence of pain, in the case of neuropathic pain, this occurs because it is one's own central or peripheral nervous system that directly receives the damage. This leads to morphological and functional changes in the sensory pathways that may become persistent without the pain disappearing. Once the damage to the nervous system is established, the natural mechanism of pain transmission is affected. This can cause pain to occur spontaneously and/or its threshold dramatically drops in such a way that the response to a pain stimulus is amplified both in amplitude and duration (hyperalgesia), or a normally harmless stimulus becomes painful (allodynia). Unlike somatic or visceral pain, when nerve damage occurs, the neural changes in susceptible individuals can be irreversible. Once established, neuropathic pain can be considered the manifestation of pathological neural plasticity that manifests itself as a state of autonomic disease of the nervous system that controls itself (von Hehn C. A., Baron, R., Woolf, C. J., 2012).
Within the etiology of neuropathic pain are physical injuries like trauma, resection or compression of the dorsal roots of the spinal cord, metabolic disorders like diabetes mellitus or vitamin B deficiency, some infections like those caused by varicella-zoster virus or HIV, neurotoxins like alcohol, or chemotherapy.
Diabetic Neuropathy
Peripheral diabetic neuropathy is a common complication of diabetes, presents itself as a variety of syndromes, among which is sensorimotor diabetic polyneuropathy (SMDPN), a very common condition affecting between 25% and 30% of diabetic patients. SMDPN is attributed to peripheral nerve damage due to metabolic and microvascular alterations as a result of chronic hyperglycemic exposure (diabetes) associated with cardiovascular risk factors (Tesfaye, S., Boulton, A. J., Dyck, P. J., et al., 2010).
Neuropathy Due to Traumatic or Postsurgical Neural Injury
When there is a mechanical injury to the peripheral nerve, the resulting pain is due to spontaneous activity generated in any site along the nociceptive pathway. However, more often, the spontaneous sensations that occur as a result of injury to the peripheral nerves are generated as a result of hyperexcitability of primary sensory neurons.
After the occurrence of a nerve injury, ectopic nervous activity is the main cause of the spontaneous sensations of pain, paresthesia or dysesthesia. The pain may be episodic or continuous, superficial or deep, and frequently presents itself as shooting pain of burning type (von Hehn, C. A., Baron, R., Woolf, C. J., 2012).
The prevalence of neuropathic pain indicates that it occurs in about 7% of the world's population (Bouhassira, D., Lanteri-Minet, M., Attal, N., Laurent, B., et al., 2008). However, the management of patients with chronic neuropathic pain is complex, and many patients do not respond to treatment, obtaining only partial pain relief, or they experience intolerable adverse effects.
For the treatment of neuropathic pain, common painkillers are generally inadequate. There is a practice that consists of giving patients pharmacological therapies at regular intervals, and effective pain treatment must be considered a favorable balance between pain relief and side effects, which not does necessarily imply having maximum analgesic effect (Vinik, A. and Casellini, C., 2013). It has been recommended to pay special attention to the following general considerations in pharmacotherapy for neuropathic pain:                For each patient, the effective and appropriate drug should be identified and its dose carefully adjusted based on its efficacy and the adverse effects it produces.        Lack of analgesic efficacy should be decided 2 to 4 weeks after treatment using an appropriate dose.        Based on the evidence of different clinical studies, any analgesic monotherapy only achieves approximately 50% of the maximum response. Therefore, it is suggested that a combination of analgesics may be very useful.Gabapentin        
Gabapentin is a structural analog of gamma aminobutyric acid (GABA), which, unlike this neurotransmitter, has an anticonvulsant effect that is not due to binding of the GABAA or GABAB receptors in the central nervous system (CNS). Gabapentin binds on the α2-δ site of the voltage-dependent calcium channels and modulates calcium input with a reduction of excitatory neurotransmission and, as a result, a decrease in the activation of the glutamate receptor and therefore a decrease in the pain signal (Dworkin, R., O'Connor, A., Audette, J., et al., 2010). So, in accordance with this mechanism of action, gabapentin, in addition to acting as an anticonvulsant, has the property of reducing the transmission of pain signals in the CNS.
The International Association for the Study of Pain has published, among others, a document for the pharmacological treatment of neuropathic pain (Attal, N. and Finnerup, N. B., 2010). In this publication, it is recommended to use specific medications that are classified into three groups: those of first line of choice, those of second line and those of third line. First line drugs are those that have been used in multiple randomized controlled clinical trials and have consistently shown their efficacy in the treatment of neuropathic pain. Among the drugs in this group is gabapentin.
Gabapentin has demonstrated efficacy in peripheral diabetic neuropathy (Finnerup, N. B., Sindrup, S. H., Jensen, T. S., 2010), in post-herpetic neuralgia (Moore, R. A., Wiffen, P., Derry, S., and McQuay, H., 2014, and Straube, S., Derry S., Moore, F. Wiffen P., and McQuay, J., 2014) and in neuropathic pain due to traumatic nerve injury (Gordh, T. E., Stubhaug, A., Jensen, T. S., et al., 2008). In the same way, in the “Treatment Guidelines for Neuropathic Pain” drawn up by the Toronto Consensus Panel, gabapentin has been classified among the first-line drugs for pain treatment in cases of peripheral diabetic neuropathy (Tesfaye, S., Vileikyte, L., Rayman, G., et al., 2011).
Lysine Clonixinate
Lysine clonixinate is an analgesic whose best known function is the inhibition of the cyclooxygenase enzymes (COX-1 and COX-2) responsible for the synthesis of prostaglandins (PGs). PGs are potent hyperalgesic mediators that modulate the signals that are transmitted along the pain pathway, increasing both transduction (peripheral sensitizing effect) and transmission (central sensitizing effect) of the pain stimulus. Therefore, inhibition of PG synthesis, both on the peripheral level and in the CNS, results in a reduction of the pain.
In addition to inhibiting the synthesis of prostaglandins, another of the effects of lysine clonixinate in the CNS that has been studied is the reduction in levels of neuronal nitric oxide synthase enzymes (NOSn) and induced enzymes (NOSi) (DiGirolamo, G., Farina, M., Ribeiro, M. L., et al., 2003). These enzymes belong to the family of nitric oxide synthases, which catalyze the production of nitric oxide (NO) from L-arginine. NO is a bioactive free radical that takes part in different physiological and pathological processes in many organs including the brain, the spinal cord and the nerves. The forms NOSn and NOSe (endothelial nitric oxide synthase) are expressed constitutively, producing NO in low concentrations. In these conditions, NO has a role in neurotransmission and vasodilatation. For its part, the expression of NOSi takes place as a response of the immune system to aggression against the body by parasites, bacterial infection, tumor growth and physical injury.
Once NOSi expression starts, this enzyme produces large quantities of NO for long periods of time, which leads to high concentrations of this and other molecules that generate high oxidizing power and severe toxicity, such as peroxynitrile, nitric dioxide and others. In particular, NO and cyclic guanosine monophosphate (cGMP) are important mediators in the neurochemical signal pathways in the spinal cord that contribute to raising the awareness of pain involved in the nociceptive process (Woolf, C. J., 2004).
Various studies have demonstrated that when peripheral nerve damage occurs, either through injury or some pathology like diabetes, the associated local inflammation and neuropathic pain that are produced are related to the production of NO by the expression of NOSi. It has also been shown that, on the level of the spinal cord, NO is involved in the development of hyperalgesia and inflammation in states of neuropathic pain (Schmidtko, A., Tegeder, I. and Geisslinger, G., 2009, and Tao, F., Tao, Y. X., Zhao, C., et al., 2004). Therefore, a reduction in iNOS levels due to the effect of lysine clonixinate could be important to reduce the progression of neuronal damage associated with neuropathic pain.
Combinations for Neuropathic Pain
For the purpose of finding the best treatment for neuropathic pain, several controlled clinical studies have been done using different medicines such as vasodilators, glutamate receptor antagonists, adrenoreceptor-α2 agonists, antidepressants and adrenergic receptor inhibitors. However, as mentioned above, the evidence indicates that only approximately 50% of the maximum response is achieved for any analgesic monotherapy, and increasing the dose is not recommended due to the increase in adverse effects. In clinical practice, 2 or more drugs are often used in combination in order to achieve some beneficial additive effect. With this objective, studies have been done using combinations of drugs for the treatment of neuropathic pain. A meta-analysis included 21 clinical studies with 1,972 participants and evaluated different combinations. It found that, of all studies included, only a comparison was possible, that is, gabapentin plus opioid versus gabapentin alone in two studies with 386 participants (Chaparro, L., Wiffen, P., Moore, R., et al., 2013). Analysis of the results of these two studies showed a modest but statistically significant superiority of the combination gabapentin plus opioid over gabapentin alone. However, this combination also produced more frequent discontinuation of the combined treatment (related to adverse effects) compared with the treatment with gabapentin alone.
The result of these and other studies is due to the fact that most of the combinations evaluated used drugs that share some effect associated with depression of the central nervous system (CNS), such as sedation or some type of cognitive dysfunction. This leads to an increase of this type of adverse effects. Consequently, an increase in the frequency of discontinuation of treatment frequently occurs, and therefore the usefulness of these combinations is very limited.
Gabapentin/Lysine Clonixinate
Given the apparent impact of the effects caused by the combination of drugs with similar profiles of adverse events, in particular in regard to CNS depression, combinations of drugs whose adverse effects are not of the same type are more favorable. In addition, the analgesic effect is increased in such a way that it is possible to reduce the content of each drug in combination compared to the drug administered separately. In particular, if medications in combination act on different sites of pain pathways or modulate different neurotransmission systems, the benefit would be to increase the level of analgesia while decreasing adverse events.
In the studies that support the present invention, it was noted that when lysine clonixinate is combined with gabapentin in specific proportions, the combination produces pharmacological effects of analgesia that indicate superadditivity (synergy) in the models of neuropathic pain associated with diabetes mellitus and by direct mechanical injury to the nerve. The foregoing makes it possible to decrease the therapeutic dose of the drugs in comparison with those that are used for each one when administered separately.
State of the Art
Patent MX 288732, “Pharmaceutical Composition Comprising a Nonsteroidal Anti-inflammatory Agent and an Anticonvulsant Agent,” describes a pharmaceutical composition composed of the combination of Gabapentin and Meloxicam (7.5 and 300 mg, respectively), in a single dosage unit, for the treatment of neuropathic pain caused by various etiologies. The description of the patent refers to studies in rats, where pain and inflammation are caused by injecting carrageenan in the leg. This pain model evaluates somatic/inflammatory pain and has been described since 1962 to validate analgesic-anti-inflammatory drugs (Winter, C. A., Risley, E. A. and Nuss, G. W., 1962). It has been used until now. In this model, the damage is not to the nerve and the pain evaluated is not of the neuropathic type. Therefore, claim number 5 of the document is not valid. Furthermore, the published patent does not include figures, drawings or tables with experimental data to show that the experiments referred to in the description were actually performed.
The publication “Evaluation of Interaction between Gabapentin and Ibuprofen on the Formalin Test in Rats” (Yoon, M., Yaksh, T., 1999) describes the combination of gabapentin and ibuprofen on a model of non-neuropathic somatic pain (injection of formalin). It also shows a solely additive effect, since the experimental DE50 (effective dose that provides an analgesic effect of 50%) did out turn out to be significantly different from the theoretical DE50, which indicates a non-synergistic additive interaction between these drugs. Furthermore, the use of this combination therapy for neuropathic pain is not mentioned or demonstrated.
U.S. Pat. No. 6,451,857 (EP 1011658), “Analgesic compositions comprising anti-epileptic compounds and methods of using same,” describes combinations of one or more antiepileptic drugs with a drug selected from the group of NMDA or NSAIDS receptor antagonists, for the relief of pain in mammals. In the description, the model of pain by injection of carrageenan is mentioned, and the examples specify the combination of gabapentin and naproxen. In the claims, pregabalin in combination is mentioned, and it does not mention that it is a combination to relieve neuropathic type pain.
The publication WO 2008/077599, “Combination Therapy of Lower Urinary Tract Disorders With α2δ Ligands and NSAIDS,” claims the combination of gabapentin and an NSAID that can be celecoxib, diclofenac, diflunisal, flurbiprofen, naproxen, nimesulide or sulindac for the monotherapy treatment of urinary incontinence. None of the claims mention the treatment of neuropathic pain.
The publication WO 2006123247 A2, “Synergistic combinations of non-steroidal antiinflammatory drugs with alpha-delta-ligands,” mentions combinations of non-steroidal anti-inflammatory drugs, particularly carprofen, with gabapentin and pregabalin for the treatment of pain and/or inflammation, particularly in dogs, cats and horses. The claims do not mention the treatment of neuropathic pain.
Experimental Design
To assess the pharmacological efficacy of the CLG combination, two experimental models in mice were used: 1) Model of pain due to diabetic neuropathy induced with streptozotocin and 2) experimental model of neuropathic pain due to injury of the sciatic nerve.
In both models, the neuropathic pain was recorded by measuring the level of mechanical allodynia, using for this purpose the Von Frey filaments assay. Later, the analgesic interaction between gabapentin and lysine clonixinate was determined through isobolographic analysis for determination of addition, antagonism or synergism of the antiallodynic effect.
Finally, the toxicity of gabapentin, lysine clonixinate and their combination was determined, through studies evaluating motor activity and neurological profile and monitoring the levels of hepatic and renal function markers.
All the experiments described below were carried out in accordance with current guidelines for the care of laboratory animals and the ethical guidelines for experimental research on pain in animals suggested by M. Zimmermann (1983).